An Oct4-Centered Protein Interaction Network in Embryonic Stem Cells

Summary Transcription factors, such as Oct4, are critical for establishing and maintaining pluripotent cell identity. Whereas the genomic locations of several pluripotency transcription factors have been reported, the spectrum of their interaction partners is underexplored. Here, we use an improved affinity protocol to purify Oct4-interacting proteins from mouse embryonic stem cells (ESCs). Subsequent purification of Oct4 partners Sall4, Tcfcp2l1, Dax1, and Esrrb resulted in an Oct4 interactome of 166 proteins, including transcription factors and chromatin-modifying complexes with documented roles in self-renewal, but also many factors not previously associated with the ESC network. We find that Esrrb associated with the basal transcription machinery and also detect interactions between transcription factors and components of the TGF-β, Notch, and Wnt signaling pathways. Acute depletion of Oct4 reduced binding of Tcfcp2l1, Dax1, and Esrrb to several target genes. In conclusion, our purification protocol allowed us to bring greater definition to the circuitry controlling pluripotent cell identity.


Supplemental Experimental Procedures
References for Oct4 interaction network      Table S1, related to Table 1  Table S1 contains the emPAI scores of interacting proteins in all Oct4 purifications.

Tables S2 to S5, related to Figure 2
Tables S2 to S5 contain the mascot scores and numbers of unique peptides of interacting proteins in all purifications of F-Sall4, F-Dax1, F-Tcfcp2l1 and F-Dax1, respectively.

Tables S6 to S9, related to Figure 2
Tables S6 to S9 contain the emPAI scores of interacting proteins in all purifications of F-Sall4, F-Dax1, F-Tcfcp2l1 and F-Dax1, respectively.

Western and RT-PCR analysis of ES cell lines
Whole cell extracts of F-Oct4 ES cells, ZHBTc4 ES cells and CGR8 ES cells were analysed by western blot with the indicated antibodies. For RT-PCR analysis, RNA was purified from 5x10 6 cells using the RNeasy protocol (Qiagen). cDNA was synthesised using 2.5μg RNA primed with random hexamers according to the Superscript First Strand Synthesis System (Invitrogen). PCRs were performed on a Roche Lightcycler by an initial denaturation at 95°C for 5 mins, followed by 45 cycles of denaturation (95°C, 5s), annealing (58°C, 10 s) and elongation (72°C, 20 s).

Mass spectrometric analysis
1D SDS-PAGE gel lanes were cut into 2-mm slices using an automatic gel slicer and subjected to in-gel reduction with dithiothreitol, alkylation with iodoacetamide and digestion with trypsin (Promega, sequencing grade), essentially as described by (Wilm et al., 1996). Nanoflow LC-MS/MS was performed on an 1100 series capillary LC system (Agilent Technologies) coupled to an LTQ-Orbitrap mass spectrometer (Thermo) operating in positive mode and equipped with a nanospray source. Peptide mixtures were trapped on a ReproSil C18 reversed phase column (Dr Maisch GmbH; column dimensions 1.5 cm × 100 µm, packed in-house) at a flow rate of 8 µl/min. Peptide separation was performed on ReproSil C18 reversed phase column (Dr Maisch GmbH; column dimensions 15 cm × 50 µm, packed in-house) using a linear gradient from 0 to 80% B (A = 0.1 % formic acid; B = 80% (v/v) acetonitrile, 0.1 % formic acid) in 70 min and at a constant flow rate of 200 nl/min using a splitter. The column eluent was directly sprayed into the ESI source of the mass spectrometer. Mass spectra were acquired in continuum mode; fragmentation of the peptides was performed in data-dependent mode. Peak lists were automatically created from raw data files using the Mascot Distiller software (version 2.1; MatrixScience). The Mascot search algorithm (version 2.2, MatrixScience) was used for searching against the NCBInr database (release NCBInr_20090222; taxonomy: Mus musculus) or the IPI_mouse_database (release 20090924). The peptide tolerance was typically set to 10 ppm and the fragment ion tolerance to 0.8 Da. A maximum number of 2 missed cleavages by trypsin were allowed and carbamidomethylated cysteine and oxidized methionine were set as fixed and variable modifications, respectively. The Mascot score cut-off value for a positive protein hit was set to 60, based on at least two peptides. In case of protein identifications with Mascot scores between 50 and 60, or that were based on only one peptide, individual peptide MS/MS spectra were checked manually and either interpreted as valid identifications or discarded. We also show a more quantitative measure of our identified proteins, emPAI score (Ishihama et al., 2005). emPAI score incorporates the number of peptides identified per protein (spectral counts) normalized by the theoretical number of peptides. This is a superior method over just counting the number of identified peptides, because it takes account of the fact that, for the same number of molecules, larger proteins and proteins with many peptides in the preferred mass range for mass spectrometry will generate more observed peptides.

Immunoprecipitation with tagged proteins
Coding sequences were amplified from mouse ES cell cDNA and inserted with an C-terminal V5-tag (Tcfcp2l1 and Zfp143) or FLAG-tag (Nac1) into a pPyCAG-driven expression vector. 46C ES cells (Ying et al., 2003) were transfected with the constructs using Lipofectamine 2000 (Invitrogen), nuclear extracts were made 24 hrs after transfection and immunoprecipitations were done, as described in the Experimental Procedures.